Isolation of Co 5'-deoxyadenosylcobinamide guanosine diphosphate

with 70% aqueous acetone. They were purified by absorption on and elutionfrom Celite, by phenol ex- traction, and finally were obtained in an aqueous ...
0 downloads 0 Views 573KB Size


Diekmann, H., Englert, G., and Wallenfels, K. (1964), Tetrahedron 20,281. Haas, E. (1936), Biochem. 2.288,123. Hope, H . (1968), Acta Cryst. (in press). Johnston, C. C., Gardner, J. L., Suelter, C. H., and Metzler, D. E . (1963), Biochemistry 2,689. Karrer, P., and Warburg, 0. (1935), Biochem. Z . 282, 157. Maas, W., Janssen, M. J., Stamhuis, E. J., and Wynberg, H. (1967), J . Org. Chem. 32,111 1. Meinhart, J. O., and Hines, M. C. (1957), Federation Proc. 16,425. Meinhart, J. O., Chaykin, S., and Krebs, E. G. (1956), J . B i d . Chem. 220,821. Rafter, G. W., Chaykin, S., and Krebs, E. G. (1953),

J . Bioi. Chem. 208,799. Segel, R., and Stein, G. (1960), J . Chem. Soc., 5254. Stamhuis, E. J., and Maas, W. (1965), J . Org. Chem. 30,2156. Stock, A., Saan, E., and Pfleiderer, G. (1961), Ann. Chem. 647,178. von Euler, H., Schlenk, F., Helwinkel, H., and Hogberg, B. (1938),2. Physiof. Chem. 256,208. Wallenfels, K., and Schuly, H . (1957), Biochem. 2. 329,75. Wallenfels, K., Hofmann, D., and Schuly, H. (1959), Ann. Chem. 621,188. Warburg, O., and Christian, W. (1934), Biochem. Z . 274,112. Wiberg,K. B. (1955), Chem. Rev. 55,713.

Isolation of Co-5'-Deoxyadenosylcobinamide Guanosine Diphosphate, a-( 2-Methyladenyl ) -Co-5'-deoxyadenosylcobamide, and Co-Methylcorrinoids from Clostridium thermoaceticum* Eckart Iriont and Lars Ljungdahlt

ABSTRACT: co-5 '-Deoxyadenosylcobinamide guanosine diphosphate (DA-GDP-cobinamide) and a-(Zmethyladenyl)-Co-5'-deoxyadenosylcobamide @A-factor A) have been found in Clostridium thermoaceticum. It is likely that the biosynthesis of complete B12 factors occurs as the c o - 5 '-deoxyadenosyl derivatives of corrinoids. DA-GDP-cobinamide, which is formed in a reaction between DA-cobinamide phosphate and guanosine triphosphate, is probably one of the intermediates in the formation of complete B1g factors. The presence


orrinoids play an important role in the metabolism of Clostridium thermoaceticum. It is likely that a-(5-methoxybenzimidazolyl)-Co-methylcobamide (methyl-factor IIIm)' and Co-methylcobyric acid are intermediates in the total synthesis of acetate from C02

2350 IRION

* From the Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106. Receiued February 5, 1968. This work was supported by a U. S. Public Health Service grant (GM 11839) from the Division of General Medical Sciences and by Contract AT (30-1)-1320 from the Atomic Energy Commission. t Present address: Farbenfabriken Bayer A. G, WuppertalElberfeld, Germany. $ Present address: Department of Biochemistry, University of Georgia, Athens, Ga. 30601. Address correspondence regarding this paper to this author. 'Abbreviations used that are not listed in Biochemistry 5 , 1445 (1966), are: factor IIIm, 5-methoxybenzimidazolylcobamide; factor A, a-2-methyladenylcobamide. AND LJUNGDAHL

of DA-factor A shows that C . thermoaceticum synthesizes B12factors with derivatives of purines as the base of the nucleotide moiety as well as of the more abundant benzimidazoles, which were reported previously. CoMethyl-factor IIIm, Co-methylcobyric acid, Co-methylcobyrinic acid pentaamide, Co-methylcobyrinic acid tetraamide, Co-methylcobyrinic acid triamide, Comethylcobinamide, and a co-methyl derivative with an unknown corrinoid moiety are present in C. thermoaceticum.

as performed by this organism (Ljungdahl et al., 1965). The high content (30-70 pmoles of corrinoids/100 g of wet cells) and the great variety of corrinoids in C . thermoaceticum provide evidence that this organism also performs the biosynthesis of BIZcompounds. We have previously reported (Irion and Ljungdahl, 1965) the presence of the complete B12 factor;2 5-methoxybenzimidazolylcobamide (factor IIIm) and of the incomplete Blz factors ; cobinamide phosphate, cobinamide, cobyric acid, and di- to pentaamides of cobyrinic acid in C. thermoaceticum. These corrinoids occur mostly as their co-5 '-deoxyadenosyl derivatives. The incomplete factors are likely intermediates in the bio2 Complete BIZfactors are defined as corrinoids with a heterocyclic base normally attached as ligand to the'cobalt atom (e.g.. 5,6-dimethylbenzimidazole in vitamin BIZ).All corrinoids lacking a base are incomplete.







synthesis of complete BI2 factors. The present communication describes the identification of c o - 5 ‘-deoxyadenosylcobinamide guanosine diphosphate (DA-GDPcobinamide), ~~-(2-methyladenyl)-Co-5 ’-deoxyadenosylcobamide (DA-factor A), and of several Co-methylcorrinoids. The presence of these compounds in C. thermoaceticum was mentioned in an earlier publication (Ljungdahl ef ai.,1966) but details of isolation and identification were not given at that time.

Acetone extraction

4 4

Purification with Celite Phenol extraction


Chromatography on carboxymethylcellulose

. 1 .

Elution with water Co-sulfitocorrinoids DA-GDP-cobinamide


Experimental Section Materials and Methods. Most methods and procedures used during this investigation are described in previous papers (Ljungdahl et a/., 1965; Irion and Ljungdahl, 1965). Samples of ~u-(2-methyladenyl)cobamide cyanide (factor A) and of cobinamide guanosine diphosphate monocyanide were generous gifts from Professor K. Bernhauer, Technische Hochschule, Stuttgart, Germany. All other chemicals are either commercially available or were obtained as described in the previous communications. Carboxymethylcellulose (Brown Co., Berlin, N. H.) was washed successively with 0.5 N sodium hydroxide, water, l N hydrochloric acid, and water until the pH was near neutrality. DEAE-cellulose (Carl Schleicher & Schuell Co.) was washed with 0.5 N sodium hydroxide and then with water until the pH was neutral. Isolation of co-5 ‘-Deoxyadenosylcobinamide Guanosine Diphosphate and ~~-(2-MethjIadenyl)-Co-5’-deoxyadenosylcobamide from C. thermoaceticum. The isolation and separation of corrinoids from C. thermoaceticum was done under dim light. The corrinoids were obtained from 200 to 500 g of wet cells by extraction with 7 0 x aqueous acetone. They were purified by absorption on and elution from Celite, by phenol extraction, and finally were obtained in an aqueous solution as described by Ljungdahl et a/. (1965). The aqueous solution was concentrated to about 5 ml in a rotary vacuum evaporator and was applied to a carboxymethylcellulose column (260 X 32 mm). The column was eluted with water to separate the corrinoids into several fractions as shown in Figure 1. The first fraction, which passed without retardation directly through the column, contained DA-GDP-cobinamide and several Co-sulfitocorrinoids. In the next 700 ml of water several minor bands were eluted, these consisted of Co-methylcorrinoids including Co-methyl-factor IIIm. DA-cobinamide phosphate was eluted next followed by DA- and Co-methylcobyrinic acid amides and several unknown corrinoids, one of which is IIa of Irion and Ljungdahl (1965). After 1800 ml of water had passed through the column two bands, one red and one brown-yellow, remained on the top of the column. The red band containing some hydroxy-B12factors was eluted with 0.04z acetic acid. The brown band was then eluted with 0.1 ammonium hydroxide. The ammonium hydroxide eluate was the major corrinoid fraction and contained DA-factor A, DA-factor IIIm, DA-cobyric acid, DA-cobinamide, and several different amides of DA-cobyrinic acid, as well as Co-


j Co-methylcobyrinic acid triamide

1 Co-methylcobyrinic acid tetraamide D


Co-methyl -factor IIIm DA-cobinamide phosphate IIA (unknown) cobyrinic acid diamide



unknown Co-methylcobyrinic acid pentaamide cobyrinic acid triamide


DA-cobyrinic acid tetraamide

K unknown

Elution with 0.04z acetic acid

-t- z

aquocorrinoids (not examined)

Elution with 0.1


DA-factor IIIm DA-factor A DA-cobinamide DA-cobyric acid DA-cobyrinic acid pentaamide Co-methylcobinamide Co-methylcobyric acid Co-methylcobyrinic acid pentaamide FIGURE 1 : Scheme for purification and chromatography on carboxymethylcellulose of corrinoids obtained by acetone extraction from C. thermoaceticum. Fraction L can be eluted with 0.5 N acetic acid instead of 0.1% ammonium hydroxide.

methylcobyric acid. The corrinoids in this fraction were extracted with phenol and then back into water. The concentrated aqueous solution was applied t o a DEAEcellulose column (80 X 14 mm) to separate the DAcobyrinic acid amides from the other corrinoids. The latter compounds were obtained in a single fraction by elution with water. The DA-cobyrinic acid amides were then eluted with water containing 5% of acetic acid. Further purification of DA-GDP-cobinamide and DA-factor A was achieved by using paper electrophoresis and paper chromatography in solvents A and B according to Irion and Ljungdahl(l965). The DA-GDPcobinamide fraction from the carboxymethylcellulose column was subjected to paper electrophoresis in 0.5 M acetic acid a t pH 2.7 to separate the weakly cationic DA-GDP-cobinamide from the anionic Co-sulfitocorrinoids. Pure DA-GDP-cobinamide was finally obtained by paper chromatography using solvent B. Paper chromatography with solvent B was also used for the separation of DA-factor A from the other corrinoids present in the aqueous eluate of the DEAE-cellulose column. DA-factor A, which moves very slowly ( R F 0.05) with solvent B, was found together with a n un-

coR R


oI Ds



C i o s t r i di u m t It e r nr o a c e f i c 11 in









z m

a 4









moving band, D2. Both bands contained radioactivity. Band D2 consisted of overlapping material from fraction C and was combined with this fraction, which was chromatographed using solvent B. Two I4C compounds, C1 and C2, were obtained. Fraction H was chromatographed with solvent B and the fastest moving corrinoid H1 was radioactive. Fraction L was also chromatographed with solvent B and the three fastest moving compounds L1, L2, and L3 were radioactive. The Co-methylcorrinoids D1, C1, C2, H1, L1, L2, and L3 were further purified by paper electrophoresis using 0.5 M acetic acid at pH 2.7 and by rechromatography with solvent B.



2352 IRION

Identijication of co-5'- Deoxyadenosylcobinamide Guanosine Diphosphate in C. thermoaceticum. The identification of DA-GDP-cobinamide is based on the following observations. Adenine and a cyanocorrinoid were formed in a reaction between the compound and KCN when the reaction was performed in the absence of light, indicating a 5 '-deoxyadenosyl moiety, which was attached to the cobalt atom. The cyanocorrinoid was identical with C1C4 previously isolated from C. known corrinoid in the band closest to the origin of the chromatogram. DA-factor A was then separated from thermoaceticum by Irion and Ljungdahl (1965). The the unidentified corrinoid IIa by the use of solvent A. absorption spectra of the isolated DA-GDP-cobinamide With this solvent DA-factor A moved somewhat more and of its mono and dicyano derivatives are shown in rapidly than the unknown compound. Figure 2. The spectrum of DA-GDP-cobinamide, which Isolation of Co-Methylcorrinoids from C. thermois unchanged between pH values 1 and 10, is similar to aceticum. Radioactive [ 14C]Co-methylcorrinoids were spectra of incomplete DA-corrinoids. Therefore, it can be concluded that the compound lacks the base of the isolated from 650 g of wet cells of C. thermoaceticum. The cells were exposed to 0.375 mmole of [14C]sodium nucleotide moiety which is coordinated with the cobalt bicarbonate (1.38 X lo9cpm) during 15 sec as described atom in complete Biz factors. The cyano derivatives of by Ljungdahl et al. (1965). The corrinoids then were the isolated compound have high absorptivity around extracted from the cells with acetone. A concentrated 275 mM which is due to the presence of the guanidine aqueous solution of the corrinoids was prepared as moiety. The spectra of the cyano compounds were indicated above and it was chromatographed on caridentical with corresponding spectra of authentic GDPboxymethylcellulose (260 X 32 mm). The column was cobinamide and the absorption maxima were those eluted with water and ten fractions, colored red-yellow, found by Ronzio and Barker (1967) for mono- and were collected separately. They contained corrinoids dicyano-GDP-cobinamide. These investigators also and were named A-K according to the sequence of synthesized a compound which they tentatively conelution as indicated in Figure 1. After elution of fraction sidered to be DA-GDP-cobinamide and the spectrum K, the column was eluted with a gradient of water to of their compound is the same as for the DA derivative 0.5 N acetic acid to obtain the remaining corrinoids in isolated from C. thermoaceticum. fraction L. Fractions A, C, D, H, and L contained The cyano derivatives of the isolated DA-GDPradioactive compounds. The radioactivity in fraction A cobinamide were compared with corresponding GDPwas associated with leucine and other amino acids, cobinamide cyanides by coelectrophoresis and cowhich apparently remained as contaminants in the chromatography on paper. The isolated compound was corrinoid fraction after purification in Celite and phenol in all instances identical with the authentic GDP-cobinextraction. Ljungdahl and Wood (1965) have shown amide cyanides. The R F values obtained during paper that radioactive amino acids are produced in pulsechromatography are listed in Table I. labeling experiments with C. thermoaceticum. The The isolated DA-GDP-cobinamide moved during other radioactive fractions contained [ 14C]Co-methylpaper electrophoresis in 0.5 N acetic acid slightly toward corrinoids, this being demonstrated by the loss of radiothe cathode ($0.1 relative to cobinamide monocyanide activity on exposure to light and the formation of [l4CC]- +l). In 0.02 M phosphate buffer (pH 7.3) the isolated methyl iodide in the reaction with iodine (Ljungdahl compound was anionic with a migration of -0.8. The et al., 1965). R F values in solvents A-C for DA-GDP-cobinamide The Co-methylcorrinoids in the radioactive fractions were all less than 0.03. The electrophoretic mobility of from the carboxymethylcellulose column were purified the monocyano derivative of the isolated GDP-cobinby paper chromatography. With solvent A, fraction D amide was -0.2 in 0.5 N acetic acid compared with separated into a faster moving band, D1, and a slower cobinamide monocyanide (f1) and for the dicyano









Paper Chromatography of GDP-cobinamide and Factor A from C. tlzermoaceticum Compared with B12.a

Electrophoretic Properties on Paper of CoMethylcorrinoids Isolated from an Acetone Extract of C. tliermouceticum.


R F Values in Solvent System Compound GDPcobinamideL Factor Ab B12









0.12 0.21

0.11 0.23

0.15 0.24


Ascending chromatography on Schleicher & Schuell paper no. 2043a at room temperature. Solvents: (D) 2-butanol saturated with 0.01% HCN, (E) 2butanol saturated with 0.01 HCN-acetic acid (lOO:l), NHI (F) 2-butanol saturated with 0.01 % HCN-25 (100 :0.5), and (G) 1-butanol-0.05 2 HCN-isopropyl alcohol-acetic acid (100: 100 :70: 1). * Authentic GDPcobinamide cochromatographed with the isolated GDP-cobinamide as did authentic factor A with the isolated factor A.

derivative -1.7 in 0.2 M phosphate buffer (pH 7.3) compared with cobyric acid dicyanide (- 1). Identification of a-(2-Methyladenyl)-Co-5’-deoxyadenosylcobamide (DA-Factor A ) in C. thermoaceticum. The isolated DA-factor A was found to be identical with the unknown IIax previously isolated from C . thernzoaceticum (Irion and Ljungdahl, 1965).The cyanocorrinoid ClC3, also previously isolated by cyanide extraction from C. thermoaceticum, was obtained together with adenine by cyanide cleavage of either IIax or of the isolated DA-factor A, showing the presence of a deoxyadenosyl moiety substituted on the cobalt atom. The absorption spectrum of the isolated DA-factor A resembles that of DA-adenylcobamide (Barker, 1962). The absorption maxima in water at pH 6.5 were at 512, 373, 318 (sh), 304, and 263 mp. The absorption at 512 mp shifted to 460 mp when the pH was lowered. This shift found in complete BI2 factors is due to the dissociation of the base from the cobalt atom. The shift occurred at about pH 5.5. The high pK value for the dissociation indicated that the base is a derivative of a purine rather than of a benzimidazole (Ladd et al., 1961). The cyano derivative of the isolated factor A cochromatographed on paper with authentic factor A with three solvent systems (Table I). The electrophoretic mobility on paper of the monocyanide of the isolated factor A was f 0 . 7 compared with +1 for cobinamide monocyanide in 0.5 M acetic acid. This basic property is also indicative of a BI2factor having a purine rather than a benzimidazole base. Finally the isolated factor A was deaminated with nitric acid according to Brown et a/. (1955). The product was chromatographed on a carboxymethylcellulose column, through which it moved with the solvent front. Addition of acetone to the concentrated aqueous eluate yielded a crystalline

I. Complete factor a-(5-Metlioxybenzimidazolyl)-Comethylcobamide (D1)

11. Incomplete factors Co-Methylcobyric acid (L2) Co-Methylcobyrinic acid pentaamide (HI) Co-Methyicobyrinic acid tetraamide (Cl) Co-Methylcobyrinic acid triamide (C2) Co-Methylcobinamide (L3) Unknown (Ll) 111. Reference compound Co-Methylcobalamin

0.5 N Acetic Acid, pH 2.7.

0.02 M Phosphate, pH 7.3


0 (neutral)


0 (neutral)

f l








f l

0 (neutral)


0 (neutral)

a Values relative to the migration of monocyanocobinamide = f l .

product which had all the properties of factor H (a-2methylhypoxanthylcobamide). Identification and Properties of Co-Methylcorrinoids from C. thermoaceticum. Co-Methylcorrinoids are present in very small amounts in C. thermoaceticum and were it not for the radioactive Co-methyl group formed from I4CO2,they probably would not have been detected. The relative specific 14Cactivities of the corrinoids were reported previously (Ljungdahl et al., 1966) together with a discussion of their role in the total synthesis of acetate from CO,. To identify the corrinoid moiety of the Co-methylcorrinoids, they were dissolved in 0.1 ml of 0.1 HCN and exposed to light. The resulting cyano-factors were identified by spectra, paper chromatography using solvents D-F, and by paper electrophoresis in 0.5 M acetic acid and in 0.2 M phosphate buffer (pH 7.3) as described by Irion and Ljungdahl (1965). It was found that D1 is Co-methyl-factor IIIm and that L2 is Comethylcobyric acid. Both compounds have already been found in C. thermoaceticum (Ljungdahl et al., 1965). The other [ 14C]corrinoidsare Co-methylcobyrinic acid triamide (C2), Co-methylcobyrinic acid tetraamide (Cl), Co-methylcobyrinic acid pentaamide (Hl), and


K I No 1D s I N


C I o Y t r i d i u in t 11 e r rn o u c e t i




TABLE 111:

Paper Chromatography of Co-MethylcorrinoidsIsolated from an Acetone Extract of C. thermoaceticum:

R F Value9 Compound




I. Complete factor a-(5-Methoxybenzimidazolyl)-Co-methylcobamide (Dl)




11. Incomplete factors Co-Methylcobyric acid (L2) Co-Methylcobyrinic acid pentaamide (Hl) Co-Methylcobyrinic acid tetraamide (Ll) Co-Methylcobyrinic acid triamide (C2) Co-Methylcobinamide (L3) Unknown (Ll)

0.18 0.10 0.04 0.02 0.26 0.15

0.22 0.28 0.32 0.46 0.25 0.18

0.20 0.12 0.08 0.04 0.28 0.13

111. Reference compound Co-Methylcobalamin




0.14 0.09c


a Ascending chromatography on Schleicher & Schuell paper no. 2043a, 20-22 hr at 22". b A = water-saturated 2-butanol; B = water-saturated 2-butanol-acetic acid (100: 1); C = 2-butanol-water-25 NH3(100: 36: 14). c These values are for red amino complexes, which are formed reversibly between incomplete factors and ammonia, when high concentration of the corrinoid is used.

Co-methylcobinamide (L3). The identification of the cobyrinic acids is not completely certain because these compounds may also be derivatives of cobinic acid amides. Finally L1 was found to be the Co-methyl derivative of a still unknown Bl2 factor in C. thermoaceticum. The electrophoretic migrations on paper of the isolated Co-methylcorrinoids in relation to monocyanocobinamide are listed in Table I1 and R F values with three different solvent systems are given in Table 111. Discussion Practically all corrinoids (98%) in C. thermoaceticum exist as co-5 '-deoxyadenosyl derivatives (Irion and Ljungdahl, 1965). This form is apparently the predominant derivative of corrinoids in nature (Barker, 1962). It is likely that the biosynthesis of complete BIZfactors starting with cobyrinic acid or one of its amides occurs in the form of the deoxyadenosyl derivatives of the corrinoids. This was discussed in a recent review by Wagner (1966). The final steps in the synthesis of complete Blz factors may involve reactions 1 and 2.


DA-cobinamide phosphate GTP -+ DA-GDP-cobinamide DA-GDP-cobinamide

2354 IRION

+ PPi

+ a-nucleoside + DA-B12factor + G M P




DA-GDP-cobinamide a-nucleoside 5'-phosphate + DA-BI2factor 5'-phosphate G M P (3)


DA-BI2factor 5'-phosphate + DA-BI2factor (2)

The a-nucleoside is probably formed by a specific trans-N-glycosidase demonstrated in Propionibacterium shermanii (Friedmann and Harris, 1965). GDP-cobin-


amide has been demonstrated in Nocardiu rugosa (Barchielli et al., 1957, 1960) and in P. shermanii (Pawelkiewicz et al., 1959). Barchielli et al. (1960) suggested that it is an intermediate in the biosynthesis of complete Bl2 factors. Our demonstration of DA-GDP-cobinamide in C. thermoaceticum supports this hypothesis. Strong evidence for this was also recently obtained by Ronzio and Barker (1967), who found that crude extracts of P. shermanii convert DA-cobinamide phosphate and GTP into DA-GDP-cobinamide. Diaquocobinamide, which lacks the deoxyadenosyl moiety, was not a substrate in this conversion. This again suggests that the biosynthesis of Blz factors normally occurs as the Co-5'deoxyadenosyl derivatives. Pawelkiewicz et al. ( 1964) have also obtained evidence for the formation of DAGDP-cobinamide in cell-free extracts of P. shermanii. An alternate pathway is possible for the formation of a DA-Bl2 factor from DA-GDP-cobinamide. Renz (1967) has isolated cyanocobalamin 5 '-phosphate from extracts of P. shermanii,and its deoxyadenosyl derivative may be involved in the biosynthesis of DA-BIPfactors (reactions 3 and 4).

+ Pi


Although deoxyadenosyl derivatives of incomplete B12factors appear to be the normal intermediates in the biosynthesis of the complete factors, corrinoids having other alkyl groups on the cobalt may also be involved







in the biosynthesis of complete factors. This was suggested by Ronzio and Barker (1967), who reported that Co-2,3-isopropylidene 5 '-deoxyadenosylcobinamide phosphate was a better substrate than DA-cobinamide phosphate in reaction 1 above. Muller and Muller (1963) have shown that Co-butylcobinamide is converted into Co-butylcobalamin by cells of P. shermanii. The presence of seven different Co-methylcorrinoids in C. thermoaceticum is an indication that Co-methyl derivatives also may be intermediates in the formation of complete factors. We reported previously (Irion and Ljungdahl, 1965) that purine cobamides were absent in C. thermoaceticum. However, with the identification of ~(2-methyladenyl)-Co-5'-deoxyadenosylcobamide as an acetone extract of C. thermoaceticum it is obvious that this organism also can synthesize purine cobamides. Brown et al. (1955) first isolated cyano-factor A from rumen intestinal bacteria and from sewage sludge. Acknowledgment The authors wish to thank Dr. Harland G. Wood for his interest in this work, which was performed in his laboratory. Added in Proof Since this article was submitted Friedmann (1968) published data showing that vitamin Blz 5'-phosphate is formed by intact cells of P. shermanii. This compound was proved kinetically and by pulse-labeling experiments t o be a precursor of vitamin BI2.Friedmann showed also that a-ribazole 5'-phosphate and GDPcobinamide were utilized in the formation of vitamin B12 5'-phosphate. These findings support the pathway shown in reactions 3 and 4 suggested in the present

paper. References Barchielli, R., Boretti, G., DiMarco, A,, Julita, P., Migliacci, A., Minghetti, A., and Spalla, C. (1960), Biochem.J. 74,382. Barchielli, R., Boretti, G., Julita, P., Migliacci, A,, and Minghetti, A. (1957), Biochim. Biophys. Acta 25,452. Barker, H. A. (1962), in Vitamin Biz und Intrinsic Faktor, 2nd European Symp., Hamburg, 82. Brown, F. B., Cain, J. C., Gant, D. E., Parker, L. F. J., and Smith, L. E. (1955), Biochem. J . 59,82. Friedmann, H. (1968), J . Biol. Chem. 243, 2065. Friedmann, H. C., and Harris, D. L. (1965), J . Biol. Chem. 240,406. Irion, E., and Ljungdahl, L. (1965), Biochemistry 4, 2780. Ladd, J. N., Hogenkamp, H. P. C., and Barker, H. A. (1961),5. Biol. Chem. 236,2114. Ljungdahl, L., Irion, E., and Wood, H. G. (1965), Biochemistry 4,2771. Ljungdahl, L., Irion, E., and Wood, H. G. (1966), Federation Proc. 25,1642. Ljungdahl, L., and Wood, H. G . (1965), J . Bacteriol. 89,1055. Muller, O., and Muller, G. (1963), Biochem. 2. 337, 179. Pawelkiewicz, J., Bartosinski, B., and Walerych, W. (1964), Ann. N . Y . Acad. Sci. 112,638. Pawelkiewciz, J., Walerych, W., and Bartosinski, B. (1959), ActaBiochem. Polon. 6,431. Renz,P. (1967), Angew. Chem. 79,311. Ronzio, R. A,, and Barker, H. A. (1967), Biochemistry 6,2344. Wagner, F. (1966), Ann. Rel;. Biochem. 35,405.


Clostridium thermoaceticum